Human papillomaviruses (HPV) encode proteins that are necessary for viral replication but also lead to the development of cancer. HPV type 16 is the most prevalent member of the group of HPVs associated with high-risk for neoplastic progression. The HPV E6 and E7 genes are selectively expressed in cervical cancers. High-risk HPV E6 proteins are best known for their ability to bind and degrade p53 and were recently reported to stimulate expression of telomerase. The HPV E6 protein is known to have p53 independent activities and to bind multiple cellular proteins. The goal of this project is to identify the functions of E6 necessary for viral genome replication in keratinocytes and compare and contrast the functions necessary for epithelial cell transformation. Aim 1 tests the hypothesis that a subset of E6 functions is necessary for HPV replication in human keratinocytes. Our approach is to clone a series of HPV 16 E6 mutations into the HPV genome and evaluate their ability to support episomal replication. Viral genome replication will be studied in monolayer and raft culture models. The mutant E6 proteins will be characterized for interactions with a subset of the E6 binding proteins and for ability to stimulate expression of hTERT, the catalytic unit of telomerase. Aim 2 tests the hypothesis that a distinct subset of E6 functions is necessary for epithelial cell immortalization and tumorigenic transformation. While E6 induced telomerase activation appears to be necessary for epithelial cell immortalization, the role of p53 is more complex. In some immortalized epithelial cells, p53 levels are normal but the integrity of the p53 pathway has not been carefully examined. We have found that cells immortalized by an E6 mutant unable to degrade p53 have a blunted response to and overcome growth inhibition induced by p14ARF. In contrast growth of hTERT immortalized cells is repressed by constitutive p14ARF expression. These data suggest that E6 may inhibit p53 in a manner distinct from inducing its degradation. In addition, the roles of most E6 binding partners in human keratinocyte immortalization and transformation have not been tested. We will critically evaluate the functions and activities of E6 required for human keratinocyte transformation with particular interest on the p14ARF-p53 and telomerase pathways, and potentially uncover other E6 connections to cell growth control.